Methods of making and using lactobacillus strains

ABSTRACT

Lactobacillus  strains that have a genetic Profile I based on Apa I, Not I, and Xba I digests are provided. Preferably, the strains decrease level of at least one of coliforms and  E. coli  within the gastrointestinal tract of an animal. A direct-fed microbial that includes the strain is additionally provided. A method of feeding an animal the strain and a method of forming a direct fed microbial that includes the strain is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/624,443, filed Jul. 22, 2003, which claims priority to U.S.Provisional Patent Application No. 60/397,654, filed Jul. 22, 2002, theentireties of both of which are incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to Lactobacillus strains for ingesting by animals.More particularly, though not exclusively, the present invention relatesto Lactobacillus strains that are useful as direct-fed microbials forpigs.

BACKGROUND OF THE INVENTION

Strains of the genus Lactobacillus are normal inhabitants of thegastrointestinal tract of many animal species. In pigs, lactobacilli areone of the principal bacterial groups in the proximal region of thedigestive tract (Barrow, P. A., R. Fuller, and N.J. Newport. 1977. Inft,Immun. 18: 586-595). Their beneficial role in the intestinal tract hasbeen attributed to their ability to survive the digestive process,attach to the epithelial lining of the intestinal tract, produce lacticacid and other antimicrobial compounds, and prevent the colonization ofpathogens via competitive exclusion (Savage, D.C. 1987. Factorsaffecting the biocontrol of bacterial pathogens in the intestine. FoodTechnol. 41: 82-87).

Many allogenic and autogenic factors influence the microbial populationof the gastrointestinal tract (Savage, D.C. 1989. Rev. sci. tech Off. inEpiz. 8: 259-273). Allogenic factors such as alterations in the diet andenvironment along with maturation of the host are major influences onthe succession of Lactobacillus strains in the gastrointestinal tract ofpigs during the post-weaning phase. Although it has been well documentedthat these changes may have severe effects on the host, little isunderstood about the distribution and diversity of lactobacilli speciesduring this period.

Current industry practices to improve health and, more specifically,reduce the levels of coliforms and E. coli within the gastrointestinaltract of pigs generally include feeding antibiotics at subtherapeuticlevels. However, the practice of feeding antibiotics to livestock hasraised concerns about increasing the antibiotic resistance of microbialpathogens in the food supply.

Another approach to improving the health of animals is to alter theinhabitants of their gastrointestinal tract. Altering the inhabitants ofthe gastrointestinal tract of animals has been attempted by feedingdirect-fed microbials to animals. The efficacy of single or multiplestrains of Lactobacillus commonly used in commercial direct-fedmicrobials has been and continues to be debated. This debate isprimarily due to inconsistent performance of previous direct-fedmicrobials. This inconsistency may be due to the fact that manycommercial direct-fed microbials are composed of Lactobacillus strainscommonly used as silage inoculants or cheese starter cultures. Thesestrains may be effective to inoculate silage or to convert milk intocheese, but have no proven efficacy as direct fed microbials for animalfeeding. While the “one strain for all products” approach may be aneconomical method for the commercial fermentation industry, this doesnot provide the best strains for each application.

In view of the foregoing, it would be desirable to provide a direct-fedmicrobial that reduces the levels of coliforms and E. coli within thegastrointestinal tract of pigs. In particular, it would be desirable toprovide a direct-fed microbial that provides a healthier intestinalmicroflora during the weaning transition period in pigs.

SUMMARY OF THE INVENTION

The invention, which is defined by the claims set out at the end of thisdisclosure, is intended to solve at least some of the problems notedabove. Lactobacillus strains that have a Profile I based on Apa I, NotI, and Xba I digests, as shown in FIG. 1 and Table 6, are provided.Preferably, the strains decrease levels of at least one of coliforms andE. coli within the gastrointestinal tract of an animal. Preferredstrains include, but are not limited to L. brevis strains, L. fermentumstrains, and L. murinus strains. Useful strains of the invention havebeen isolated from the pars oesophagea of a pig. A particularlypreferred strain is L. brevis strain 1E-1, although any Lactobacillusstrain having a Profile I based on Apa I, Not I, and Xba I digests, asshown in FIG. 1 and Table 6 are expected to work in the invention.

A method of feeding an animal is also provided. The method comprisesfeeding the animal a Lactobacillus strain that has a Profile I based onApa I, Not I, and Xba I digests, as shown in FIG. 1 and Table 6.Preferably, the strain decreases levels of at least one of coliforms andE. coli within the gastrointestinal tract of an animal.

A direct-fed microbial is additionally provided. The direct-fedmicrobial includes at least one Lactobacillus strain that has a ProfileI based on Apa I, Not I, and Xba I digests, as shown in FIG. 1 and Table6. The direct-fed microbial additionally includes a carrier.

Also provided is a method of forming a direct fed microbial. In themethod, a culture is grown in a liquid nutrient broth. The cultureincludes at least one Lactobacillus strain that has a Profile I based onApa I, Not I, and Xba I digests, as shown in FIG. 1 and Table 6. Thestrain is separated from the liquid nutrient broth.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred exemplary embodiments of the invention are illustrated in theaccompanying drawings.

FIG. 1 shows Apa I, Not I, and Xba I digests of various strains from pig1, including strain 1E-1.

FIG. 2 is a graph showing mean coliform for pre-weaning pigs 9-13 daysold.

FIG. 3 is a graph showing mean E. coli for pre-weaning pigs 9-13 daysold.

FIG. 4 is a graph showing mean coliform for weaning pigs 19-23 days old.

FIG. 5 is a graph showing mean E. coli for weaning pigs 19-23 days old.

FIG. 6 is graph showing mean coliform populations for pre-weaning pigs.

FIG. 7 is graph showing mean coliform populations for weaning pigs.

FIG. 8 is graph showing mean E. coli populations for pre-weaning pigs.

FIG. 9 is a graph showing mean E. coli populations for weaning pigs.

FIG. 10 is a graph showing ileal villus height/crypt depth ratios forpigs at 10, 22, and 28 days.

FIG. 11 is a graph showing duodenum villus height/crypt depth ratios forpigs at 10, 22, and 28 days.

FIG. 12 is a graph showing the number of sulfuric goblet cells in theduodenum of pigs on d 10, 21, and 28 of age (interaction, P<0.06). Meanswithin each day post-weaning with different letter designations differsignificantly (P<0.06).

Before explaining embodiments of the invention in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or being practiced or carriedout in various ways. Also, it is to be understood that the phraseologyand terminology employed herein is for the purpose of description andshould not be regarded as limiting.

DETAILED DESCRIPTION

In accordance with the present invention, there may be employedconventional molecular biology and microbiology within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Described herein are Lactobacillus strains that have positive effects onthe health of animals. Preferred Lactobacillus strains will now bedescribed that are useful in pigs. This example is not intended to limitthe invention to Lactobacillus strains usable only in pigs. TheLactobacillus strains of the invention are isolated from an animal, suchas a pig. Lactobacillus strains of the invention preferably reduce thelevels of coliforms and E. coli within the gastrointestinal tract ofpigs. The Lactobacillus strains also provide a healthier intestinalmicroflora during a pre-weaning period in pigs. Thus, the Lactobacillusstrains provide a healthier intestinal microflora during a pre-weaningand weaning period in pigs.

Lactobacillus strains of the invention have a profile I based on Apa I,Not I and Xba I digests, as shown in FIG. 1 and Table 6 (below).Preferred Lactobacillus strains include, but are not limited to, L.brevis, L. fermentum, and L. murinus. A preferred Lactobacillus brevisstrain is 1E-1, which was isolated from the intestinal tract of ahealthy, weaned pig. Strain 1E-1 is available from the microorganismcollection of the American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. 20110, under accession number PTA-6509, and wasdeposited on Jan. 12, 2005.

The Lactobacillus strains of the invention can be used as a direct-fedmicrobial. In a preferred embodiment, the direct-fed microbial is L.brevis strain 1E-1. Furthermore, the multiple Lactobacillus strains canbe combined as a direct-fed microbial.

Characterization and Screening of Lactobacillus Strains:

In one exemplary evaluation of the bacteria of the present invention,the intestinal tracts of thirteen weaned pigs, ten healthy and threewith scours, were sampled for the Lactobacillus strains found therein.As is detailed in Example 1 below, twenty-five numerically dominantisolates were selected from samples of the pars oesophagea, duodenum,jejunum, and ileum from each pig.

Isolates were identified using biochemical and carbohydrate fermentationtests. Plasmid profiling and pulsed-field gel electrophoresis (PFGE)were used to attempt to distinguish between strains of lactobacilliwithin a species. Higher numbers of lactobacilli were detectedthroughout the intestinal tract of healthy pigs when compared to theintestinal tract of sick pigs. The highest lactobacilli counts for bothhealthy and sick pigs were found in the pars oesophagea samples (healthypigs-1.3×10⁸ CFU/g and sick pigs-1.6×10⁶ CFU/g). The lowest counts werefound in the jejunal samples for both groups (healthy pigs-1.9×10⁶ CFU/gand sick pigs-4.3×10⁵ CFU/g).

Biochemical identification of the isolates indicated that thelactobacilli populations of healthy pigs were much more homogeneous thanlactobacilli populations of sick pigs. In healthy pigs, the majority ofthe isolates were identified as L. brevis. Depending on the location, L.brevis accounted for 35-90% of the lactobacilli population. Similar, butnot always, identical plasmid profiles were observed among L. brevisisolates. Identical plasmid profiles were observed among isolatesidentified as different species. PFGE was useful in identifyingindividual strains within a species.

Preparation and Feeding of Lactobacillus Strains:

A direct-fed microbial of the invention includes a Lactobacillus strainthat has a Profile I based on Apa I, Not I and Xba I digests, as shownin FIG. 1 and Table 6. A preferred strain is the L. brevis strain 1E-1,although other Lactobacillus strains having a Profile I can be used. Acarrier can be added to the direct-fed microbial. The carrier can be aliquid carrier, a solid carrier, or any other suitable carrier. Apreferred liquid carrier is a milk replacer. Milk replacers aretypically milk substitutes in powdered form that are mixed with water toform a composition that resembles milk. Another preferred liquid carrieris water. Dry carriers include, but are not limited to, animal feed.

The Lactobacillus strains of the present invention may be presented invarious physical forms, for example, as a top dress, as a water solubleconcentrate for use as a liquid drench or to be added to a milk replaceror gels. In a preferred embodiment of the top dress form of theLactobacillus strains, a freeze-dried Lactobacillus strain fermentationproduct is added to a carrier, such as whey, limestone (calciumcarbonate), rice hulls, yeast culture, dried starch, or sodium silicoaluminate.

In a preferred embodiment of the water soluble concentrate for a liquiddrench or milk replacer supplement, a freeze-dried Lactobacillus strainfermentation product is added to a water soluble carrier, such as whey,maltodextrin, sucrose, dextrose, dried starch, or sodium silicoaluminate, and a liquid is added to form the drench or the supplement isadded to milk or a milk replacer. In a preferred embodiment of the gelsform, a Lactobacillus strain fermentation product is added to a carrier,such as one or more of vegetable oil, sucrose, silicon dioxide,polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid,and ethoxyquin to form the gel. An artificial coloring can be added tothe gel.

Particularly preferred ways of feeding the direct-fed microbial includea milk supplement (replacer) fed during lactation 7-19 days prior toweaning, a single dose of a gel paste or drench given 1-2 days prior toweaning followed by dosing in water systems in the nursery for 7 days,and a single does of a gel paste or drench given 1-2 days prior toweaning followed by dosing in gruel feed in the nursery for 2-3 days.The direct-fed microbial can be fed in other forms, for differingperiods of time, and at different stages in the pig's weaning.

Typically, the direct-fed microbial is formed by growing a cultureincluding the Lactobacillus strain of choice in a liquid nutrient broth.The Lactobacillus strain of the culture is then separated from theliquid nutrient broth, such as by centrifugation. The Lactobacillusstrain can then be freeze-dried. The freeze-dried Lactobacillus straincan be added to a carrier. This addition can be done immediately or at asubsequent time. Where the freeze-dried Lactobacillus strains are addedat a subsequent time, they are preferably stored in a waterproofcontainer, such as a foil pack.

The Lactobacillus strains of the invention can be fed to an animal. In apreferred embodiment, the animal is fed a Lactobacillus brevis strain1-E1. Particularly useful results have been obtained when young pigs,including pre-weaning pigs, weaned pigs, and post-weaned pigs, are fedone or more the Lactobacillus strain of the invention.

Preferably, the animal is fed the Lactobacillus strain such that theamount of Lactobacillus strain delivered to the animal is about 1×10⁸CFU to about 1×10¹⁰ CFU per day. More preferably, the animal is fed theLactobacillus strain such that the amount of Lactobacillus straindelivered to the animal is about 5×10⁹ CFU per day. However, it shouldbe noted that higher and lower doses of the Lactobacillus strain can befed to the animal and are believed to have a positive effect on theanimal.

As is shown below in detail, feeding the Lactobacillus strain of theinvention to animals altered the intestinal flora of the animals,decreasing levels of coliforms and E. coli in the animals. Maintaining anormal healthy intestinal microflora during the profound environmentaland nutritional changes at weaning is critical to ensure optimalperformance for pigs. Feeding the Lactobacillus strain of the inventionalso increased the average daily gain, increased the villus:crypt ratioin the animals, and decreased the number of sulfuric goblet cells, as isalso shown in detail below.

Effect of Feeding Lactobacillus Strain 1E-1 on Pre-weaning andPost-weaning Pigs:

The effects of feeding L. brevis strain 1E-1 on the gastrointestinalmicroflora of pre-weaning, weaning, and post-weaning pigs have beendetermined. As is detailed below in Example 2, sows and gilts wererandomly assigned to one of three treatments. In Example 2, four littersreceived no milk replacer (control), five litters received milkreplacer, and five litters received milk replacer supplemented withstrain 1E-1.

In Example 2, coliforms and E. coli were enumerated from parsoesophageal, duodenal, jejunal, and ileal regions of intestinal tractsfrom one pig per litter at 9-13 days of age (pre-weaning) and at 19-23days of age (weaning). In pre-weaning pigs, E. coli and coliformpopulations in pars oesophageal, duodenal, and ileal regions ofpre-weaning pigs were not significantly different in the three groups.As is shown in more detail in Example 2, pigs receiving strain 1E-1 hadsignificantly lower jejunal E. coli populations compared to control(P<0.02) and milk replacer (P<0.05). Jejunal coliform populations tendedto be lower for pigs receiving strain 1E-1 compared to control pigs(P<0.12) but were not significantly different compared to pigs receivingmilk replacer. There were no treatment effects on populations ofcoliforms and E. coli in the pars oesophageal and duodenal regions forpigs at weaning. Pigs receiving strain 1E-1 had significantly lower E.coli populations in the jejunal region compared to control (P<0.01) andmilk replacer (P<0.11). There were no significant treatment effects onjejunal coliform populations for pigs at weaning. In the ileal region ofweaning pigs, the coliform populations neared significance for pigsreceiving strain 1E-1 when compared to control (P<0.07). E. colipopulations were significantly lower for pigs receiving strain 1E-1compared to control pigs (P<0.05) and pigs receiving milk replacer(P<0.02). These results show that feeding strain 1E-1 provides ahealthier intestinal microflora during lactation.

The use of L. brevis strain 1E-1 is shown herein to reduce the levels ofcoliforms and E. coli within the gastrointestinal tract of pigs,providing a healthier intestinal microflora during the pre-weaningperiod. The strongest response shown by strain 1E-1 was in the distalregions of the gastrointestinal tract more than the proximal regions,within pigs at weaning (19-23 days old) more than the pre-weaning pigs(9-13 days old), and against E. coli more than coliforms.

In addition, feeding the Lactobacillus strain to animals improves thehealth of the animal. For instance, feeding the Lactobacillus strain toyoung pigs decreases the incidence of scours in the young pigs.

Additionally, as is detailed below in Example 3, populations of E. coliand coliforms in the small intestine were reduced pre- and post-weaningwhen pigs were supplemented with 1E-1.

Intestinal morphology improved when animals were fed the Lactobacillusstrain of the invention. For instance, villus:crypt ratio was greaterand the number of sulfuric goblet cells was less with supplementationwith the Lactobacillus strain. These data indicate that supplementingwith strain 1E-1 and other strains having a Profile I based on Apa I,Not I and Xba I digests, as shown in FIG. 1 and Table 6 pre-weaningimproves nursery performance and provides a healthier intestinalenvironment. An increase in weight at weaning in pigs fed theLactobacillus strain was also observed.

EXAMPLES

The following Examples are provided for illustrative purposes only. TheExamples are included herein solely to aid in a more completeunderstanding of the presently described invention. The Examples do notlimit the scope of the invention described or claimed herein in anyfashion.

Example 1 Characterization of the Predominant Lactobacilli Isolated fromthe Intestinal Tract of Post-Weaned Pigs Materials and Methods

Pigs:

Thirteen crossbred pigs raised in a commercial facility in Arkansas wereused in this study. After weaning at 21 days, pigs were fed a complexPhase 1 prestarter diet. At 7-10 days post-weaning, 3-5 pigs wereselected from either a healthy group or a group of pigs identified ashaving scours and transported to Oklahoma State University. Pigs werekilled by exsanguation and samples of the pars oesophagea, duodenum,jejunum, and ileum were aseptically removed along with 25 g samples offecal and stomach contents. Three repetitions of this procedure werecompleted for a total sample size of 13 pigs (10 healthy and 3 withscours).

Isolation and Maintenance of Cultures:

Pars oesophagea, duodenal, jejunal, and ileal sections were washed with20 ml of sterile buffer (0.3 mM KH₂PO₄, 1 mM MgSO₄, 0.05% cysteinehydrochloride, pH 7.0) and cut open with surgical scissors to expose theepithelial lining. To remove cells from intestinal tissue, 22 g sampleswere placed in sterile bags and agitated in a stomacher for 60 sec.Stomach and fecal contents were suspended in sterile buffer and mixed bystomaching. Lactobacilli populations were enumerated on LBS agar. Plateswere incubated anaerobically (GasPak) for 48 h at 37° C. Twenty-fiveisolated colonies from the highest dilution of each tissue sample werepicked into 10 ml tubes of MRS (Difco, Detroit, Mich.) broth. Strainswere routinely propagated in MRS broth (Difco, Detroit, Mich.) at 37° C.and stored in MRS broth containing 10% glycerol at −75° C.

Biochemical Screening of Isolates:

Isolates picked from LBS plates were confirmed as lactobacilli by theGram-stain reaction, cell morphology, and catalase reaction. The speciesidentity of isolates was determined using API Rapid CH kits (AnalytabProducts, Plainview, N.Y.) according to the manufacturer's directions.Fermentation patterns were observed and recorded for each isolate at 24,36, and 48 h. Fermentation patterns of each isolate were compared todifferential characteristics provided in Bergey's Manual for speciesidentification.

Plasmid DNA Isolation:

Plasmid DNA was isolated from the lactobacilli strains as follows: a 1%inoculum taken from a 24 hour culture was placed into 10 ml of sterileMRS broth and incubated at 37° C. until the optical density (660 nm)reached 0.8 (log phase). Cell suspensions were then harvested bycentrifugation (12,000×g for 15 min). The supernatant was decanted andthe pellet resuspended in 1 ml of Tris-EDTA buffer containing 15%sucrose. Resuspended cells were stored in 1.5 ml centrifuge tubes at−20° C. until plasmid DNA analysis was performed. Frozen samples wereallowed to thaw at room temperature. Cells were washed by harvesting(12,000×g for 5 minutes) and resuspending the pellet in 1 ml ofTris-EDTA buffer containing 15% sucrose. After washing, the pellet wasresuspended to a final volume of 250 μl with fresh Tris-EDTA-sucrosebuffer and mixed well by vortexing. Lysozyme (50 μl of a 60 mg/mlsolution) was added, and the tubes were incubated on ice for 1 hour.Pronase (10 mg/ml: pre-incubated at 37° C. for 1 hour) was added (35 μl)followed by incubation at 37° C. for 30 minutes.

Following incubation, 0.25 M EDTA was added (111 μl) to the sample andthe tubes held for 15 minutes on ice. Tris-EDTA containing 20% SDS wasadded (111 μl) and held on ice for an additional 15 minutes. Sodiumacetate (75 μl of a 3.0 M solution) was added followed by a 30 minuteincubation on ice. Debris was pelleted by centrifugation (12,000×g for15 minutes) and the supernatant transferred to a clean 1.5 mlmicrocentrifuge tube. Cold ethanol (750 μl of 95%) was added to the tubecontaining the supernatant and mixed well by gently inverting the tubeseveral times. The samples were stored at −20° C. for 1 hour toprecipitate the DNA. DNA was pelleted by centrifugation (12,000×g for 15minutes) and allowed to dry. The DNA was resuspended in 40 μl Tris-EDTAbuffer, 5 μl of tracking dye was added and the mixture loaded onto anagarose gel. DNA was separated by gel electrophoresis using a 0.7%agarose gel at 50 volts. Agarose gels were examined after a 45 minutestaining period in ethidium bromide solution.

Preparation of Intact Genomic DNA:

Intact genomic DNA from representative strains was isolated from cellsembedded in agarose beads using a modification of the method ofRehberger, T. G. 1993. Curr. Microbiol. 27: 21-25. Cultures were grownto mid-log stage in MRS broth, harvested by centrifugation (9,000×g for10 min), and resuspended to one-tenth the original volume in ET buffer(50 mM EDTA, 1 mM Tris-HCl, pH 8.0). The cell suspension was mixed withan equal volume of 1% low-melting point agarose (Beekman Instruments,Palo Alto, Calif.), loaded into a syringe and injected into tygon tubingwhere it was allowed to solidify. The solidified cell-agarose mixturewas forced into cold ET buffer and gently vortexed to break the stringinto smaller bead like pieces. The beads were resuspended in 10 ml of10×ET buffer containing 5 mg/ml of lysozyme and incubated on ice for 2hours to digest the cell wall material.

After incubation, the beads were harvested by centrifugation (4,000×gfor 10 min) and resuspended in 10 ml of lysis buffer (10×ET buffercontaining 100 ug/ml of proteinase K and 1% Sarkosyl), followed byincubation at 55 C for 5-7 hours to lyse the cells and release thegenomic DNA. After cellular lysis, the beads were harvested bycentrifugation (4,000×g for 10 min), resuspended in 10 ml of 1 mMphenylmethylsulfonyl fluoride, and incubated at room temperature for 2hours to remove contaminating protease activity. The beads containingthe purified DNA were washed three times in TE buffer (10 mM Tris-HCl, 1mM EDTA-Na₂, pH 7.5), resuspended in 10 ml of TE buffer and stored at 4°C. until restriction endonuclease digestion.

In Situ Restriction Endonuclease Digestion and Pulsed GelElectrophoresis:

Agarose beads containing DNA were equilibrated in 1× restrictionendonuclease buffer for 1 hour before enzyme digestion. After the beadswere equilibrated, 10-20 units of the restriction enzyme were added to90 ul of beads and incubated at the appropriate temperature for 6-8hours. Following digestion, the enzymes were inactivated by heating for10 minutes at 65° C. This melted the beads and allowed for easy loadingonto the gel for fragment separation.

DNA fragments were separated on 1.0% agarose gels in 0.5×TBE buffer at15° C. for 20 hours using a CHEF-DRIII electrophoresis system (Bio-Rad,Hercules, Calif.). Each set of restriction endonuclease digests wereseparated at different initial and final pulse times to provide maximumseparation of small, medium, and large fragments. To determine themolecular size of the DNA fragments lambda DNA multimers, intact yeastchromosomes and restriction fragments of lambda DNA were included asstandards.

Results and Discussion

Higher mean numbers of lactobacilli populations were detected in allgastrointestinal samples from healthy pigs compared to pigs with scours(Table 1). However, the variation in the lactobacilli populations amonghealthy pigs for all sample locations was greater than the differenceseen between healthy and sick pigs.

TABLE 1 Populations of lactobacilli in the digestive tract ofpost-weaning pigs. Lactobacillus populations^(a) Location^(b) Pig NumberE D J I S F Healthy 1 6.61 4.64 3.82 4.08 5.69 8.86 2 6.04 5.44 4.723.90 6.08 5.20 3 7.76 5.83 6.44 5.67 8.63 9.30 4 8.23 5.52 6.86 6.467.67 9.74 5 8.73 7.72 6.11 6.63 8.98 9.97 6 7.14 5.08 5.89 6.18 8.149.28 7 8.58 6.34 6.63 8.52 8.26 8.61 8 7.99 5.72 5.50 5.20 8.04 9.50 96.99 5.94 6.18 7.20 7.50 8.72 10  7.41 5.66 6.08 5.59 8.44 9.65 Scour13  5.98 4.93 4.08 4.04 6.96 4.81 14  6.41 6.88 6.00 6.64 7.26 9.20 15 6.08 4.99 5.44 5.62 6.64 7.80 ^(a)Log10 lactobacilli per ml or g ofsample. ^(b)Symbols: E = pars oesophagea, D = duodenum, J = jejunum, I =ileum, S = stomach contents, F = feces.

Independent of the health of the animal, differences in lactobacillipopulations were observed among different regions in thegastrointestinal tract. The par oesophageal region of all animalscontained the highest number of lactobacilli compared to othergastrointestinal regions. The jejunal region of all animals containedthe lowest number of lactobacilli compared to other gastrointestinalregions.

Identification of the predominant Lactobacillus species from thedigestive tract samples is shown in Table 2. In some cases, thepredominant species accounted for 100% of the total lactobacillipopulation. L. brevis and L. murinus were found to be the most commonpredominant species in healthy pigs while L. plantarum and L. murinuswere found to be most common in pigs with scours.

TABLE 2 Identification of the predominant Lactobacillus species fromdigestive tract samples of representative pigs. Pig Number Location^(a)Major species^(b) Percentage^(c) Healthy 1 E L. brevis 57 D L. brevis 95J L. brevis 50 I L. brevis 100 2 E L. brevis 40 D L. brevis 100 J L.brevis 80 I L. brevis 60 3 E L. murinus 63 D L. murinus 80 J L.fermentum 30 I L. murinus 44 7 E L. murinus 80 D various strains 50 J L.brevis 82 I L. acidophilus 59 10 E L. plantarum 50 D L. murinus 70 J L.murinus 90 I L. murinus 60 Scour 13 E L. plantarum 56 D L. plantarum 64J L. plantarum 70 I L. murinus 60 14 E L. fermentum 60 D L. brevis 32 JL. murinus 80 I L. murinus 56 ^(a)Symbols: E = pars oesophagea, D =duodenum, J = jejunum, I = ileum ^(b)as determined by carbohydratefermentation patterns ^(c)percent of the total lactobacilli from eachlocation for each pig

The predominant Lactobacillus species identified for each region of thedigestive tract (Table 3) was found to be different between healthy andsick pigs. L. brevis was found to be the most common predominant speciesin three regions of healthy pigs while L. plantarum and L. murinus werefound to be most common in two regions each of pigs with scours.

TABLE 3 Predominant Lactobacillus species isolated from differentregions of the digestive tract. Health Status Location^(a) Majorspecies^(b) Percentage^(c) Healthy E L. brevis 39 L. murinus 44 D L.brevis 52 L. murinus 35 J L. brevis 45 I L. brevis 43 Scour E L.plantarum 26 L. fermentum 30 D L. plantarum 32 J L. murinus 55 L.plantarum 35 I L. murinus 55 L. plantarum 40 ^(a)Symbols: E = parsoesophagea, D = duodenum, J = jejunum, I = ileum ^(b)as determined bycarbohydrate fermentation patterns ^(c)mean percentage of totallactobacilli from all pigs

The predominant Lactobacillus species identified from each pig examined(Table 4) indicated that L. brevis was the predominant species in 3 ofthe 5 healthy pigs and L. plantarum and L. murinus were the predominantspecies in pigs with scours.

TABLE 4 Predominant Lactobacillus species isolated from representativepigs. Pig Number Major species^(a) Percentage^(b) Healthy 1 L. brevis 762 L. brevis 69 3 L. murinus 49 7 L. brevis 37 10 L. murinus 60 Scour 13L. plantarum 59 14 L. murinus 30 ^(a)as determined by carbohydratefermentation patterns ^(b)mean percentage of total lactobacilli from alllocations

Plasmid profiling was used in an attempt to distinguish strains oflactobacilli. Strains were assigned to a plasmid profile type forcomparison to other strains from different regions and pigs. Table 5lists the seven major profile types observed in this study. All plasmidprofiles types were found to be common to two or more pigs and two ormore regions. However, no profile type was shared among healthy and sickpigs. Fewer number of isolates were examined (140) for plasmids fromsick pigs, which may have affected this observation. Plasmid profiletype I was the most common profile in healthy pigs, while type III wasmost common in pigs with scours. Plasmid profiling was not asdiscriminatory a typing technique as genomic DNA fingerprinting todistinguish between strains within a species.

TABLE 5 Identification of the predominant plasmid profiles. PlasmidNumber of Profile Plasmids Molecular weights (kb) Examples I 7 2.5, 2.7,3.1, 13.6, 71.5 2D-4, 7J-3, 10J-8 II 6 3.2, 6.2, 7.4, 32.1 2D-8, 2D-17,7J-6 III 2 3.4, 3.7 13I-2, 13J-8, 13D-16 IV 3 3.9, 14.2 14J-1, 14J-4,14J-7 V 4 2.5, 3.5, 4.2, 22.6 7E-2, 7E-4, 7E-6 VI 4 3.5, 4.5, 20.2 7E-8,7E-9, 7E-10 VII 0 N/A N/A no example of profile

Comparisons of genomic DNA fingerprints produced by restrictionendonuclease digestion of intact genomic DNA were used to determine thegenetic relatedness among strains (data not shown). In general, amajority of strains isolated from the same animal were found to haveidentical Apa I, Not I, and Xba I fingerprints. Populations oflactobacilli in different gastric regions were composed of similarstrains. To date, no evidence was found indicating distinct populationsfor the different regions of the gastrointestinal tract examined in thisstudy. In contrast, distinct populations were identified that werespecific for healthy and sick pigs. These findings indicate a distinctdifference in the dominant strains of the lactobacilli populationsbetween healthy and sick pigs.

Pulsed-field gel electrophoresis was useful at identifying differencesamong phenotypically indistinguishable strains. As an example, at leastthree different L. brevis strains and three different L. murinus havebeen identified from the isolates examined from pig 1 (Table 6). Inaddition, genomic fingerprints have also been found to be identicalbetween phenotypically different strains. This may be due to geneticchanges in the genes responsible for the carbohydrate fermentation(s)found to distinguish the strains biochemically. These changes could haveresulted in the loss of function but may not have altered therestriction sites or the distances between them and therefore, goundetected as differences by genomic fingerprints.

TABLE 6 Genomic restriction endonuclease digestion profiles oflactobacilli from representative pigs Genomic digestion profile^(a)Strain Biochemical identification Pig 1 I E-1 L. brevis D-3 L. fermentumD-5 L. brevis D-15 L. brevis D-22 L. brevis J-3 L. brevis J-6 L. murinusJ-8 L. murinus I-5 L. brevis I-14 L. brevis I-17 L. brevis I-24 L brevisII E-7 L. brevis E-11 L. murinus E-17 L. brevis E-25 L. brevis III J-19L. murinus J-20 NA IV E-23 L. brevis Pig 13 V E-1 NA E-2 L. plantarumE-3 L. murinus E-6 L. agilis E-8 L. plantarum D-1 L. plantarum D-2 L.plantarum D-3 L. agilis D-7 L. agilis J-1 L. plantarum J-3 L. murinusJ-4 L. plantarum J-5 L. plantarum J-8 L. murinus VI D-6 L. sake D-8 L.sake VII J-2 L. murinus ^(a)Based on APA I, Not I and Xba I digestsNA—No identifiable species

Example 2 Influence of Lactobacillus brevis Strain 1E-1 on theGastrointestinal Microflora and Performance of Pre-Weaning and WeaningPigs

From the study described in Example 1, it was determined that theintestinal tract of healthy pigs had higher levels of lactobacilli.Genetic analysis of the lactobacilli found in healthy pigs indicated ahomogenous population of strains, whereas the lactobacilli populationsfound in the sick pigs were heterogeneous. The majority of the isolates(59%) were identified as a single genotype (Profile I based on Apa I,Not I and Xba I digests) that was biochemically identified as L. brevis.This strain is now referred to as 1E-1 and is the Lactobacillus strainused in this example. Lanes 1 and 14 contain a Lambda concatamer as amolecular weight (MW) marker. FIG. 1 shows Apa I digests (left handlane), Not I digests (middle lane), and Xba I digests (right hand lane)of various strains, including strain 1E-1. All Lactobacillus strainswith a Profile I based on Apa I, Not I, and Xba I digests are expectedto work in the invention.

Materials and Methods

Sows and gilts were blocked by parity and sire and randomly allotted toone of three treatments as they were placed in the farrowing room at110-112 days of gestation. Litters, starting at birth, received nosupplemental milk replacer (control), supplemental milk replacer (18.5%solids, 1.5 lbs/gallon) without 1E-1 (milk), or supplemental milkreplacer (18.5% solids, 1.5 lbs/gallon) with 1E-1 (milk plus 1E-1). Pigsreceived treatments up to the day of weaning. At weaning, pigs withineach group were ranked by weight. A phase I diet, fed for the first twoweeks post-weaning, contained 3.75% spray-dries plasma and at least 15%lactose. A phase II diet, fed until the completion of the study (28days), contained 1.0% plasma, 1.5% blood cells, and at least 8% lactose.

The milk replacer system used in this study was an in-line system. Themilk replacer was supplied to the pigs ad libitum in a small bowlsupplied by a central 30-gallon tank. The tank was equipped with a hydropump and a pressure regulator that pumped the milk replacer to the pensas needed. A baby pig nipple inside each bowl allowed milk to flow intothe bowl only when touched by a pigs nose. This was used to minimizespillage and waste of the milk replacer. The entire system was flushedon a daily basis with hot water to remove spoiled milk or sediment, andfresh milk was prepared using a commercial milk replacer (Merrick'sLitter-Gro, Union Center, Wis.).

Whole intestinal tracts were removed from one randomly selected pig perlitter at 9-13 days of age and from another at 19-23 days. Tracts wereimmediately placed in a Whirl-pak® bag containing approximately 200 mlsterile phosphate buffer (0.3 mM KH₂PO₄, 1 mM MgSO₄, 0.05% cysteinehydrochloride, pH=7.0). Tracts were sent to Agtech Products, Inc. forfurther analysis, which included enumerating E. coli and coliforms,harvesting the bacterial community in the tracts, and community DNAisolation to trace 1E-1 throughout the tracts.

Whole tracts were aseptically cut into pars oesophageal, duodenal,jejunal, and ileal sections, and each section was rinsed with sterilephosphate buffer until all contents were washed out. The section was cutlengthwise to expose the epithelial lining, and the sterile rinse wasrepeated. The weight of each section was recorded and the section placedin a new Whirl-pak® bag. Sterile phosphate buffer (99 ml) was added toeach bag and masticated for 60 seconds. Each sample was plated on VRB(Difco Sparks, Md.) for the enumeration of coliforms and CHROMagar(CHROMagar Paris, France) for the enumeration of E. coli. Spiral platingtechniques were used at 10⁻¹ and 10⁻³ dilutions on the Autoplate 4000(Spiral Biotech, Inc., Norwood, Mass.). The remaining liquid was pouredinto a sterile 250 ml centrifuge bottle and the intestinal scrapingsharvested by centrifugation at 8000 rpm for 15 minutes. The supernatantwas carefully removed and the pellet was resuspended with 10 ml MRS+10%glycerol. The sample was then transferred to a sterile 15 ml Falcon®tube and stored at −20° C. until DNA isolation.

DNA was isolated from the harvested cells using a High Pure PCR TemplatePreparation Kit (Roche Diagnostics GmbH, Mannheim, Germany). 1E-1 wasthen isolated within the community DNA by using PCR on the 16S-23Sintergenic spacer region of each sample. Specific primers were used thatannealed to conserved regions of the 16S and 23S genes(Tilsala-Timisjarvi, A., and T. Alatossava. 1997 Development ofoligonucleotide primers from the 16S-23S rRNA intergenic sequences foridentifying different dairy and probiotic lactic acid Lactobacillusstrain by PCR. Int. J. Food Microbiol. 35: 49-56). PCR was performedfollowing the procedures of Tannock et al. (1999. Identification ofLactobacillus isolates from the gastrointestinal tract, silage, andyoghurt by 16S-23S rRNA gene intergenic spacer region sequencecomparisons. 65: 4264-4267). PCR mixtures contained 5 μl of 10×polymerase buffer (Boehringer Mannheim), 200 μM each deoxynucleosidetriphosphate, 80 pM each primer, 8 μl DNA, and 2.6 U of Expand HighFidelity PCR System (Boehringer Mannheim GmbH, Mannheim, Germany) DNApolymerase in a total volume of 50 μl. The PCR program began with apre-incubation at 94° C. for 2 min., then 95° C. for 30 s, 55° C. for 30s, and 72° C. for 30 s. This was repeated for 30 cycles and finishedwith a 5-min incubation at 72° C. PCR products were then isolated byelectrophoresis in a 1% agarose gel and visualized by UVtransillumination after being stained in ethidium bromide solution.

Results Gastrointestinal Microflora:

Pre-Weaning Pigs: There were no significant treatment effects onpopulations of coliforms within the pars oesophageal, duodenal, andileal sections of pre-weaning pigs (FIG. 2). The jejunal coliformpopulations tended to be lower for pigs receiving milk replacer plus1E-1 compared to control pigs (P<0.12) but were not significantlydifferent when compared to pigs receiving milk replacer alone.

E. coli populations enumerated within the pars oesophageal, duodenal,and ileal sections were not significantly different among pre-weaningpigs receiving the three treatments (FIG. 3). However, in the ilealsection, there was a trend for lower levels of E. coli in the pigsreceiving milk plus 1E-1 compared to control pigs and those receivingmilk replacer alone. Pigs receiving milk plus 1E-1 had significantlylower jejunal E. coli populations compared to control (P<0.02) and milkreplacer alone (P<0.05).

Weaning Pigs: The coliform levels within the pars oesophageal, duodenal,and jejunal regions of weaning pigs showed no significance betweentreatments (FIG. 4). Coliform levels in the ileal region, however,tended to be lower for pigs receiving milk replacer plus 1E-1 whencompared to control (P<0.07), but were not significantly different whencompared to milk replacer alone.

The E. coli levels in the weaning pigs showed some large differencesbetween treatments within the distal regions of the gastrointestinaltract (FIG. 5). There were no significant treatment effects on E. colilevels within the pars oesophageal region. In the duodenum, E. colilevels tended to be lower for pigs receiving milk replacer plus 1E-1compared to control (P<0.13). Within the jejunal region, pigs receivingmilk replacer plus 1E-1 had significantly lower E. coli populationscompared to the control (P<0.01) and the difference in E. colipopulations was nearing significance compared to milk replacer alone(P<0.11). The E. coli populations in the ileal region showed asignificant reduction in E. coli levels for pigs receiving milk replacerplus 1E-1 when compared to control (P<0.05) and milk replacer alone(P<0.02).

Overall, the microbial data showed a reduction in coliforms and E. colifor pre-weaning and weaning pigs receiving milk replacer plus 1E-1. Themost noticeable response was in the distal regions of thegastrointestinal tract compared to the pars oesophageal and duodenal(proximal) regions. The reduction in E. coli levels in the pigs wasgreater than the reduction in the level of coliforms. A greaterdifference was also observed between treatments in pigs at weaningcompared to pre-weaning pigs.

The establishment of strain 1E-1 in the gastrointestinal tract wasstudied using the 16S-23S intergenic spacer region of 1E-1 (Tannock, G.W., et al. 1999. Identification of Lactobacillus isolates from thegastrointestinal tract, silage, and yoghurt by 16S-23S rRNA geneintergenic spacer region sequence comparisons. Appl. Environ. Microbiol.65: 4264-4267). The intergenic spacer region has been known to be highlyvariable between lactobacilli. Tannock's results indicated that this wasa relatively simple and rapid method by which lactobacilli can beidentified without resorting to the use of species-specific primers. Theresults of this analysis, however, showed identical PCR products betweennative lactobacilli and strain 1E-1. Even a Cfo I restriction digest didnot distinguish between these isolates. Therefore, the use of the16S-23S intergenic spacer region has not been useful for tracing strain1E-1 within the gastrointestinal tract.

Performance Data:

Suckling Pigs:

The suckling performance data is shown below in Table 7. Pigs receivingstrain 1E-1 had a significant increase in average daily gain (ADG) frombirth to weaning when compared to the control (P=0.07), but notsignificantly different when compared to pigs fed milk replacer alone.At five days to weaning and ten days to weaning, pigs receiving strain1E-1 showed a significant increase in ADG when compared to control(P=0.05 and P=0.06, respectively), but not when compared to milkreplacer alone. There was no significant difference in weight at weaningfor pigs receiving any of the treatments.

TABLE 7 Suckling pig performance data Item Control Milk Milk + 1E-1 ADG,g ADG, birth to 5 d 128    163    138    ADG, birth to 10 d 166   186    182    ADG, birth to weaning 192^(b)   240^(a,b)   262^(a)   ADG,5 d to 10 d 204    208    226    ADG, 5 d to weaning 214^(d)  267^(c,d)   305^(c)   ADG, 10 d to weaning 221^(f)   299^(e,f) 347^(e)   Weight, kg Litter birth weight 14.72 12.28 14.33 Weight at day5 20.39 21.74 20.74 Weight at day 10 28.41 21.74 28.18 Weight atweaning* 39.55 44.93 43.69 *Adjusted for age at weaning (average 19.64d) ^(a,b)Means with differing superscripts are significantly different;P = 0.07 ^(c,d) Means with differing superscripts are significantlydifferent; P = 0.05 ^(e,f)Means with differing superscripts aresignificantly different; P = 0.06

Nursery pigs:

The nursery pig performance data is shown below in Table 8. Nursery pigperformance was monitored to determine the long-term effects of thetreatments. In phase 1, the pigs receiving strain 1E-1 had a significantincrease in ADG when compared to the pigs receiving milk replacer alone(P<0.05), but were not significantly different compared to pigs fed nomilk (control). In phase 2, a significant improvement in the gain:feedratio was observed for pigs receiving milk alone compared to controlpigs (P<0.05), however, there was no significant difference whencompared to pigs receiving strain 1E-1. Overall (phase 1 and 2combined), there was no significant difference for ADG or gain:feedamong the treatments. Pigs fed strain 1E-1 had a significantly higherweight at the end of phase 1 compared to pigs receiving milk replaceralone (P=0.07), but the weight was not significantly higher compared tocontrol. At the end of Phase 2, the pigs receiving milk plus strain 1E-1tended to have a higher weight when compared to control pigs (P=0.11),but the weight was not different compared to the milk replacer alone.

TABLE 8 Nursery pig performance data Item Control Milk Milk + 1E-1 Phase1 ADG, g   239^(a,b)  211^(b)  258^(a) ADFI, g 228 211 250 Gain: Feed    1.109     1.031     1.139 Phase 2 ADG, g 466 487 517 ADFI, g 633 620673 Gain: Feed     0.736^(b)     0.790^(a)      0.769^(a,b) Phase 1-2ADG, g 352 355 388 ADFI, g 431 435 461 Gain: Feed     0.823     0.828    0.850 Weight, kg Initial    5.21    5.50    6.31 Phase 1    8.51^(c,d)     8.26^(d)     9.88^(c) Phase 2    15.03^(e)   15.19^(e,f)   17.12^(f) ^(a,b)Means with differing superscripts aresignificantly different; P < 0.05 ^(c,d)Means with differingsuperscripts are significantly different; P = 0.07 ^(e,f)Means withdiffering superscripts are significantly different; P = 0.11

Example 3 Influence of Lactobacillus brevis Strain 1E-1 on Performance,Intestinal Microflora, and Intestinal Morphology of Pre-Weaning andWeaning Pigs Materials and Methods

In each experiment, litters were allotted to two treatments atfarrowing: either a control milk supplement, or the control milksupplement containing strain 1E-1. The milk supplement contained 18.5%solids. Treatments were administered throughout the lactation period.After weaning, pigs were grouped 6 pigs per pen.

Coliforms and E. coli were enumerated from pars oesophageal, duodenal,jejunal, and ileal regions of the enteric tracts, and gut morphology wasassessed from one pig/litter at approximately 10 (pre-weaning) and 22(weaning) days of age, and after weaning at 28 days of age. Gutmorphology was examined to determine villus height and area, crypt depthand the different mucins (neutral, acidic, and sulfuric) produced fromenteric goblet cells. Duodenum and ileum tissue samples were taken andsectioned at 4-6 μm. Sections were mounted on polylysine-coated slidesand stained with 1) hematoxylin and eosin, 2) alcian blue and periodicacid schiff, and 3) high iron dye. Intestinal microflora populationswere determined for pars oesophageal, duodenal, jejunal, and ilealsections. Samples from these regions were processed and each sample wasplated. VRB was used for enumeration of coliforms. CHROMagar was usedfor enumeration of E. coli. Spiral plating techniques were used at 10⁻¹and 10⁻³ dilutions on an Autoplate 4000.

Results and Discussion

Growth Performance:

Strain 1E-1 supplementation did not affect pig growth performance duringthe pre- or post-weaning periods. The lack of a performance responsewith strain 1E-1 supplementation in this Example is likely a consequenceof lower coliform and E. coli populations in all regions of thegastrointestinal tract from all ages of pigs examined in thisexperiment. In Example 3, coliform and E. coli levels in pre-weaningpigs ranged from 100 to 1000 times lower than coliform and E. colilevels in pigs from Example 2. At weaning, coliform and E. coli levelsin pigs from Example 3 were 1000 times lower in the proximal regions ofthe gastrointestinal tract and 10 to 100 times lower in the distalregions compared to pigs in Example 2. Although the coliform and E. colilevels were reduced by feeding L. brevis 1E-1 in Example 3, pigperformance was not significantly improved due to decreased pathogenicchallenge.

Intestinal Microflora:

Pigs receiving strain 1E-1 had lower jejunal coliform populationspre-weaning (P <0.15) and at weaning (P<0.07) compared to pigs providedonly milk supplement, as is shown in FIGS. 6 and 7, respectively. Inaddition, pigs receiving strain 1E-1 had lower (P<0.001) ileal coliformpopulations at weaning compared to pigs provided only milk supplement,as is shown in FIG. 7. FIG. 8 shows that pigs receiving strain 1E-1 hadlower jejunal E. coli populations pre-weaning (P<0.13). FIG. 9 showsthat strain 1E-1 reduced E. coli populations at weaning in the jejunal(P<0.06) and ileal (P<0.001) compared to pigs provided only milksupplement. In sum, strain 1E-1 was shown to reduce levels of coliformsand E. coli within the gastrointestinal tract of pigs, providing ahealthier intestinal microflora during the post-weaning period.

Intestinal Morphology: As is shown in FIG. 10, pigs provided strain 1E-1had greater (P<0.01) ileal villus:crypt ratio at 10 days of age comparedto control pigs, although there was no difference at 22 and 28 days ofage (interaction, P<0.05). The greater ileal villus:crypt ratioindicates that strain 1E-1 increases the maturation of the distal regionof the gut in 10 day old pigs. FIG. 11 shows that pigs provided strain1E-1 had greater (P<0.01) duodenum villus:crypt ratio at 22 days of agecompared to control pigs, although there was no difference at 10 and 28days of age (interaction, P<0.02).

The number of duodenal sulfuric goblet cells was less (P=0.06) when pigswere provided strain 1E-1 compared to control pigs at 10 days of age,although there was no difference at 22 and 28 days of age (interaction,P=0.06; FIG. 12). Sulphomucins are normally absent from the smallintestine, but can be produced by crypt goblet cells when the smallintestinal mucosa is altered (Specian, R. D. and M. G. Oliver. 1991.Functional biology of intestinal goblet cells. Am. J. Physiol.260:C183-C193). The decrease in the number of duodenal sulfuric gobletcells by strain 1E-1 in pigs at 10 days of age may be an indication of ahealthier gastrointestinal tract. In addition, the lower number ofsulfuric goblet cells, combined with the increase in villus:crypt ratioin strain 1E-1-supplemented pigs suggests that strain 1E-1 affords someprotection from the intestinal disruption that occurs at weaning.

It is understood that the various preferred embodiments are shown anddescribed above to illustrate different possible features of theinvention and the varying ways in which these features may be combined.Apart from combining the different features of the above embodiments invarying ways, other modifications are also considered to be within thescope of the invention. The invention is not intended to be limited tothe preferred embodiments described above, but rather is intended to belimited only by the claims set out below. Thus, the inventionencompasses all alternative embodiments that fall literally orequivalently within the scope of the claims.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. A method of forming a direct fed microbial, the method comprising:(a) growing, in a liquid nutrient broth, a culture including at leastone Lactobacillus strain that has a Profile I based on Apa I, Not I andXba I digests, as shown in FIG. 1 and Table 6; and (b) separating thestrain from the liquid nutrient broth.
 17. The method of claim 16,wherein the Lactobacillus strain comprises L. brevis strain 1E-1 ATCCAccession No. PTA-6509.
 18. The method of claim 16, further comprisingfreeze-drying the strain.
 19. The method of claim 18, further comprisingadding the freeze-dried strain to a carrier.
 20. The method of claim 16,wherein the Lactobacillus strain comprises a brevis strain having all ofthe identifying characteristics of the L. brevis strain 1E-1 ATCCAccession No. PTA-6509.
 21. The method of claim 19, wherein the carrieris selected from the group consisting of: whey, limestone, rice hulls,yeast culture, malodextrin, sucrose, dextrose, dried starch and sodiumsilico aluminate.
 22. The method of claim 16, wherein the Lactobacillusstrain comprises at least one of a L. brevis strain, a L. fermentumstrain, and a L. murinus strain.
 23. The method of claim 22, wherein theLactobacillus strain comprises a L. fermentum strain.
 24. The method ofclaim 22, wherein the Lactobacillus strain comprises a L. murinusstrain.
 25. The method of claim 16, further comprising adding a carrierto the separated strain of step (b).
 26. The method of claim 25, whereinthe carrier is a liquid carrier or a solid carrier.
 27. The method ofclaim 26, wherein the liquid carrier is a milk replacer.
 28. The methodof claim 26, wherein the solid carrier is animal feed.
 29. The method ofclaim 16 further comprising using the separated strain of step (b) as awater soluble concentrate.
 30. The method of claim 16 further comprisingusing the separated strain of step (b) as a top dress.
 31. The method ofclaim 16 further comprising using the separated strain of step (b) as agel.
 32. The method of claim 31, wherein the gel comprises a carrier.33. The method of claim 32, wherein the carrier is selected from thegroup consisting of: vegetable oil, sucrose, silicon dioxide,polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid,and ethoxyquin.
 34. The method of claim 32, wherein the gel furthercomprises an artificial coloring agent.